Project 2 will validate a model of human-like TB latency in New Zealand White rabbits, and apply this model to test interventions against latent TB infections (LTBl). The world's latently infected population comprises the reservoir which feeds the global tuberculosis TB epidemic. Novel approaches to treat latent infections would contribute greatly to TB eradication. There is a critical need to develop an animal model that reflects the human spectrum of latency. The mouse is a poor model for LTBl; and our preliminary data and work by others suggests that latently-infected non-human primates are more likely percolators with significantly more bacterial replication and mutational stress than is seen with LTBl in humans. Rabbits infected with Mycobacterium tuberculosis (Mtb) strain CDC1551 develop a primary infection that is rapidly contained, after which the Mtb bacilli soon become undetectable in all rabbit tissues. Yet, like human latency, low numbers of Mtb bacilli persist in an undetectable (latent) state because they can be reactivated by immune suppression with corticosteroids. We will more fully study the range of bacterial activity that occurs in human latency at our clinical site in Vitoria, Brazil. These results will be used to benchmark, and if needed, fine tune (modify) our rabbit latency model. Next, we will use infections with a high-density bar-code Mtb library to measure (for the first time) the rate of Mtb cell killing during latency, the number of clones that survive during LTBl, and the replication and mutation rate during this disease state. We will then use high-density bar-coded Mtb to map the pathway of clonal re-emergence during reactivation. Mtb gene knockouts will be used to identify bacterial factors essential for latency LTBl. The rabbit latency model, bar-coded library and latency knockouts will then be used as tools to evaluate the efficacy of new therapeutic interventions in eradicating paucibacillary infection. Correlates of immunological protection identified in human TB in Project 1 will be queried in these efficacy studies. Cross-fertilizing with Project 4, we will establish the overlap between bacterial factors required for persistence vs. latency. The discovery of novel latency and persistence genes will identify new druggable targets active against the latent state.